Amplification of HIV-1 sequences for detection of sequences associated with drug-resistance mutations

ABSTRACT

Sequences of nucleic acid oligonucleotides for amplifying different portions of gag and pol genes of HIV-1 and for detecting such amplified nucleic acid sequences are disclosed. Methods of amplifying and detecting HIV-1 nucleic acid in a biological sample using the amplification oligonucleotides specific for gag and pol target sequences are disclosed.

RELATED APPLICATION

[0001] This application claims priority to provisional U.S. applicationNo. 60/229,790, filed Sep. 1, 2000, which is incorporated by reference,and claims the benefits available under 35 U.S.C. § 119(e).

FIELD OF THE INVENTION

[0002] This invention relates to diagnostic detection of HumanImmunodeficiency Virus (HIV-1), and more particularly relates tocompositions and assays for detecting HIV-1 nucleic acid sequences usingnucleic acid amplification and detection of amplified sequences,particularly sequences related to development of HIV-1 drug resistance.

BACKGROUND OF THE INVENTION

[0003] HIV-1 is the causative agent of acquired immunodeficiencysyndrome (AIDS). Early detection of HIV-1 infection is important fordetermining effective treatment of the infection and to avoidtransmission of the infectious virus in body fluids, even before theinfected individual manifests symptoms. Early detection of the presenceof HIV-1 nucleic acid sequences in infected tissue or body fluid canlead to earlier treatment and steps to prevent spread of the virus toothers. To be effective in early diagnosis, reagents and procedures todetect HIV-1 nucleic acid sequences must be able to detect relativelylow numbers of viral copies in the tested sample (e.g., a few hundredcopies per ml of plasma). Furthermore, diagnostic methods that provideadditional information about the HIV-1 present in an infectedindividual, such as the HIV-1 subtype and/or mutational changesassociated with viral drug-resistance, are useful for prognosis.

[0004] Drug-resistance mutations (substitutions, deletions or insertionsof one or more nucleic acid bases) have been found in HIV-1 patients whohave been treated with drugs and have experienced a resurgence of HIV-1proliferation and symptoms. Such mutations, which result in a phenotypicchange whereby the virus becomes resistant to an antiretroviral drug,have found in the gag gene, often affecting protease cleavage sites, andthe pol gene, in both the protease and the reverse transcriptase (RT)coding regions (Gingeras et al., 1991, J. Infect. Dis. 164(6):1066-1074;Richman et al., 1991, J. Infect. Dis. 164(6):1075-1081; Schinazi et al.,1993, Antimicrob. Agents Chemother. 37(4):875-881; Najera et al., 1994,AIDS Res. Hum. Retroviruses 10(1 1):1479-1488; Eastman et al., 1995, J.AIDS Hum. Retrovirol. 9(3):264-273; Frenkel et al., 1995, J. Clin.Microbiol. 33(2):342-347; Shirasaka et al., 1995, Proc. Natl. Acad. Sci.USA 92(6):2398-2402; Leal et al., 1996, Eur. J. Clin. Invest.26(6):476-480; Cleland et al., 1996, J. AIDS Hum. Retrovirol.12(1):6-18; Schmit et al., 1996, AIDS 10(9):995-999; Vasudevechari etal., 1996, Antimicrob. Agents Chemother. 40(11):2535-2541; Winslow etal., 1996, AIDS 10(11):1205-1209; Fontenot et al., Virology 190(1):1-10;Cornelissen et al., 1997, J. Virol. 71(9):6348-6358; Ives et al., 1997,J. Antimicrob. Chemother. 39(6):771-779).

[0005] Information on viral mutations present in an HIV-1 infectedpatient can be used by a clinician to determine appropriate treatment,such as whether to begin or maintain the patient on a particularantiretroviral therapy. Moreover, continued testing of patient samplesthat permits characterization of the HIV-1 during treatment can indicateemergence of a drug-resistant virus, thereby allowing the clinician toalter the therapy to one that is more effective. Evidence suggestingthat drug resistance testing has clinical utility comes fromretrospective and prospective intervention-based studies (Durant et al.,1999, Lancet 353:2195-2199; Clevenbergh et al., 1999, Antiviral Ther. 4:Abstract 60; Baxter et al., 1999, Antiviral Ther. 4: Abstract 61; Cohenet al., 2000, 7^(th) Conference on Retroviruses and OpportunisticInfections (San Francisco, Calif.), Abstract 237). Some mutations knownto confer drug resistance affect 20 codons of the protease codingsequence and 27 codons of the reverse transcriptase (RT) coding sequence(Hirsch et al., 2000, JAMA 18:2417-2426). A comprehensive list of 190mutations in pol was reported by Schinazi et al. (Int'l Antiviral News8:65-91 (2000)). Mutations that affect gag cleavage sites have beenshown to compensate for loss of enzyme activity due to resistancemutations in protease. Thus, there is a need for genotypic assays thatprovide sequence information on relevant codons, because such assays maydetect a viral mutant which could contribute to drug failure, even if itis a minor component of the patient's viral population.

[0006] The HIV-1 genome is highly variable, with three groups (M, O andN) described based on their genetic relatedness. The most prevalentgroup, M, contains subtypes (A to J), with subtypes A, B and Caccounting for about 95% of the viral subtypes found worldwide. SubtypeE, which is frequently found in Asia, is a recombinant virus thatincludes subtype A sequence in the gag and pol genes. Therefore, aneffective diagnostic assay must be able to detect at least one of the A,B and C subtypes, and, preferably, all of them.

[0007] Detection of HIV-1 by using a variety of assays and reagents hasbeen described previously. For example, U.S. Pat. Nos. 5,594,123,5,176,995 and 5,008,182 (Sninsky et al.) disclose detection based on thepolymerase chain reaction (PCR) to amplify HIV-1 nucleic acid. U.S. Pat.No. 5,688,637 (Moncany et al.) discloses oligonucleotide primersequences for selected regions of HIV -1 genes, and methods ofamplifying viral sequences using the primers and detecting the amplifiedproducts. U.S. Pat. Nos. 5,712,385 (McDonough et al.) and 5,856,088(McDonough et al.) disclose amplification oligonucleotides, probes andmethods for detecting HIV-1 sequences. U.S. Pat. No. 5,786,177 (Moncanyet al.) discloses methods of amplifying HIV-1 nucleotide sequences forgene expression and purification of the polypeptides produced. HIV-1nucleic acid sequences useful for detecting the presence of HIV-1 havebeen disclosed in U.S. Pat. No. 5,773,602 and EP 0 178 978 (Alizon etal.), U.S. Pat. Nos. 5,843,638 (Montagnier et al.), 6,001,977 (Chang etal.), 5,420,030 (Reitz et al.) and 5,869,313 (Reitz et al.), and EP 0181 150 (Luciw et al.). PCT No. WO 9961666 discloses a method fordetecting polymorphic mutations in HIV genetic sequences which providean indication of an increased risk of an imminent viral drug-resistancemutation.

[0008] Methods of amplifying nucleic acids to produce more copies of atarget sequence have been described previously. For example, U.S. Pat.Nos. 4,683,195, 4,683,202, and 4,800,159 (Mullis et al.) disclose PCRamplification which uses a thermocycling series of denaturation andpolymerization reactions to produce many copies of a target sequence.Amplification methods that rely on transcription using an RNA polymerasehave been disclosed in U.S. Pat. Nos. 5,399,491 and 5,554,516 (Kacian etal.), U.S. Pat. No. 5,437,990 (Burg et al.), PCT Nos. WO 8801302 and WO8810315 (Gingeras et al.), U.S. Pat. No. 5,130,238 (Malek et al.); andU.S. Pat. Nos. 4,868,105 and 5,124,246 (Urdea et al.). The ligase chainreaction (LCR) uses four different oligonucleotides to amplify a targetand its complementary strand by using cycles of hybridization, ligation,and denaturation (EP No. 0 320 308). Strand displacement amplification(SDA) uses a primer that contains a recognition site for a restrictionendonuclease that nicks one strand of a hemimodified DNA target duplex,followed by primer extension and strand displacement steps (U.S. Pat.No. 5,422,252 (Walker et al.)).

[0009] Nucleic acid sequences may be detected by using hybridizationwith a complementary sequence (e.g., oligonucleotide probes) (see U.S.Pat. Nos. 5,503,980 (Cantor), 5,202,231 (Drmanac et al.), 5,149,625(Church et al.), 5,112,736 (Caldwell et al.), 5,068,176 (Vijg et al.),and 5,002,867 (Macevicz)). Hybridization detection methods may use anarray of probes on a DNA chip to provide sequence information about thetarget nucleic acid which selectively hybridizes to an exactlycomplementary probe sequence in a set of four related probe sequencesthat differ one nucleotide (see U.S. Pat. Nos. 5,837,832 and 5,861,242(Chee et al.)).

SUMMARY OF THE INVENTION

[0010] According to one aspect of the invention, there is provided anucleic acid oligomer for amplifying a nucleotide sequence of HIV-1,comprising a sequence selected from the group consisting of SEQ ID NO:5to SEQ ID NO:22 and SEQ ID NO:33 to SEQ ID NO:68. In one embodiment, thenucleic acid oligomer has a nucleic acid backbone that includes one ormore 2′-O-methoxy linkages, peptide nucleic acid linkages,phosphorothioate linkages, methylphosphonate linkages or any combinationof these linkages. In another embodiment, the oligomer is apromoter-primer having a sequence selected from the group consisting ofSEQ ID NO:5 to SEQ ID NO:10 and SEQ ID NO:33 to SEQ ID NO:45, wherein a5′ portion of the sequence includes a promoter sequence for T7 RNApolymerase. Another embodiment is a mixture of nucleic acid oligomersthat includes oligomers for amplifying a first gag sequence and having anucleotide sequence selected from the group consisting of SEQ ID NO:5,SEQ ID NO:11, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:47,SEQ ID NO:17 and SEQ ID NO:59. Another embodiment is a mixture ofnucleic acid oligomers includes oligomers for amplifying a second gagsequence and having a nucleotide sequence selected from the groupconsisting of SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:34, SEQ ID NO:36, SEQID NO:48, SEQ ID NO:49, SEQ ID NO:18, and SEQ ID NO:60. Another mixtureembodiment includes oligomers for amplifying a Protease sequence andhaving a nucleotide sequence selected from the group consisting of SEQID NO:7, SEQ ID NO:13, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:19, SEQ ID NO:61, and SEQ ID NO:62. One mixture ofoligomers includes oligomers for amplifying a first reversetranscriptase (RT) sequence and having a nucleotide sequence selectedfrom the group consisting of SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54,SEQ ID NO:20, SEQ ID NO:63, SEQ ID NO:64 and SEQ ID NO:65. Anotherembodiment is a mixture that includes oligomers for amplifying a secondRT sequence and having a nucleotide sequence selected from the groupconsisting of SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:42, SEQ ID NO:43, SEQID NO:55, SEQ ID NO:56, SEQ ID NO:21, and SEQ ID NO:66. Another mixtureincludes oligomers for amplifying a third RT sequence and having anucleotide sequence selected from the group consisting of SEQ ID NO:10,SEQ ID NO:16, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:57, SEQ ID NO:58,SEQ ID NO:22, SEQ ID NO:67, and SEQ ID NO:68.

[0011] Another aspect of the invention is a labeled oligonucleotide thatspecifically hybridizes to an HIV-1 sequence derived from gag or polsequences, having a base sequence selected from the group consisting ofSEQ ID NO:23 to SEQ ID NO:29, and a label that results in a detectablesignal. In one embodiment, the labeled oligonucleotide includes in itsnucleic acid backbone one or more 2′-O-methoxy linkages, peptide nucleicacid linkages, phosphorothioate linkages, methylphosphonate linkages orany combination these linkages. In another embodiment, the labeledoligonucleotide includes a label that is a compound that produces aluminescent signal that can be detected in a homogeneous detectionsystem. In one embodiment, the label is an acridinium ester (AE)compound and the oligonucleotide hybridizes to an HIV-1 sequence derivedfrom gag sequences and has a base sequence selected from the groupconsisting of SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25. In anotherembodiment, the label is an AE compound and the oligonucleotidehybridizes to an HIV-1 sequence derived from pol sequences and has abase sequence selected from the group consisting of SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, and SEQ ID NO:29.

[0012] Another aspect of the invention provides a method of detectingHIV-1 in a biological sample. The method includes the steps of providinga biological sample containing HIV-1 nucleic acid; mixing the samplewith two or more amplification oligomers that specifically amplify atleast one HIV-1 target sequence contained within gag and pol sequencesunder conditions that allow amplification of nucleic acid, wherein theamplification oligomers have sequences selected from the groupconsisting of:

[0013] SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:17, and SEQ ID NO:59 to amplify a firstgag sequence;

[0014] SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:18, and SEQ ID NO:60 to amplify a secondgag sequence;

[0015] SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:50, SEQ ID NO:51, SEQ ID NO:19, SEQ ID NO:61 and SEQ ID NO:62 toamplify a first pol sequence, which is a protease sequence;

[0016] SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:41, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:20, SEQ IDNO:63, SEQ ID NO:64, and SEQ ID NO:65 to amplify a second pol sequence,which is a first reverse transcriptase (RT) sequence;

[0017] SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:55, SEQ ID NO:56, SEQ ID NO:21, and SEQ ID NO:66 to amplify a thirdpol sequence, which is a second RT sequence; and

[0018] SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:22, SEQ ID NO:67, and SEQ ID NO:68 toamplify a fourth pol sequence, which is a third RT sequence, or acombination of oligomers selected from these groups that allowsamplification of at least one gag sequence and at least pol sequence;amplifying the target sequence to produce an amplified nucleic acidproduct; and detecting the presence of the amplified nucleic acidproduct. In one embodiment, the amplifying step uses atranscription-mediated amplification method which is conducted insubstantially isothermal conditions. In one embodiment, the detectingstep uses a labeled oligomer having the sequence of SEQ ID NO:23, SEQ IDNO:24 or SEQ ID NO:25, or a mixture of these oligomers, to hybridizespecifically to the amplified nucleic acid produced from a gag sequence;a labeled oligomer having the sequence of SEQ ID NO:26, SEQ ID NO:27,SEQ ID NO:28 or SEQ ID NO:29, or a mixture of these oligomers, tohybridize specifically to the amplified nucleic acid produced from a polsequence; or a mixture of at least two labeled oligomers, wherein themixture comprises one or more first labeled oligomers selected from thegroup consisting of SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, andone or more second labeled oligomers selected from the group consistingof SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29 tohybridize specifically to the amplified nucleic acid produced from atleast one gag and at least one pol sequence. Another embodiment of themethod has a detecting step that detects hybridization of the amplifiednucleic acid to an array of nucleic acid probes. In another embodiment,the method may also include the step of contacting the sample containingHIV-1 nucleic acid with at least one capture oligomer having a sequencethat hybridizes specifically to the HIV-1 nucleic acid, thus forming ahybridization complex that includes the HIV-1 nucleic acid, andseparating the hybridization complex from other sample components.

[0019] The accompanying drawings illustrate some embodiments of theinvention. The drawings, together with the description, serve to explainand illustrate the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 is a schematic drawing of HIV-1 gag and pol genetic regions(shown left to right in a 5′ to 3′ orientation at the top) showing therelative positions and sizes of amplified sequences (Gag1, Gag2, Prt,RT1, RT3 and RT4, shown below the gag and pol regions) that are producedusing the amplification oligonucleotides and methods of the invention.In one embodiment, the upper group of the Gag1, Gag2 and RT3 regions areamplified in one multiplex reaction, and the lower group of the Prt, RT1and RT4 regions are amplified in another multiplex reaction.

[0021]FIG. 2 is a schematic drawing of HIV-1 gag and pol regions andamplified sequences as described in FIG. 1, but in this embodiment, theupper group of the Gag1, Prt and RT3 regions are amplified together inone multiplex reaction, and the lower group of the Gag2, RT1 and RT4regions are amplified in another multiplex reaction.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention includes methods and oligonucleotides todetect HIV-1 nucleic acid in a biological sample, by amplifying one ormore HIV-1 target regions and detecting the amplified sequences. Theamplified target regions are ones that include many codons that maypotentially be mutated when an HIV-1 virus becomes drug resistant. Thedetection step may be performed using any of a variety of known ways todetect a signal specifically associated with the amplified targetsequence, such as by hybridizing the amplification product with alabeled probe and detecting a signal resulting from the labeled probe.The detection step may also may provide additional information on theamplified sequence, such as all or a portion of its nucleic acid basesequence.

[0023] The compositions and methods of the present invention are usefulfor detecting the presence of HIV-1 sequences and providing additionalinformation about the infective agent, such as its genetic subgroup ordrug-resistance phenotype based on detectable sequence information.Thus, the invention provides useful diagnostic and prognosticinformation on an HIV-1 infection to a health provider and the patient.In preferred embodiments, multiple different portions of the HIV-1genome are amplified in a multiplex reaction to produce multiple copiesof different HIV-1 sequences in a single reaction vessel. The amplifiedproducts from different regions are then detected and/or analyzedfurther. Multiplex reactions minimize the number of individual reactionsthat are performed for a sample and, because multiple regions areamplified, multiplex reactions avoid the potential of a false negativeresult if one region were insufficiently amplified. In one embodiment,the method amplifies multiple different portions of the HIV-1 genomeusing amplification oligonucleotides in two different multiplexreactions to cumulatively amplify about 2.5 kb of HIV-1 sequence.

[0024] The methods of the present invention preferably includeamplification using an isothermal transcription-mediated nucleic acidamplification method, as previously disclosed in detail in U.S. Pat.Nos. 5,399,491 and 5,554,516 (Kacian et al.). The methods include adetection step that may use any of a variety of known methods to detectthe presence of nucleic acid by hybridization to a probeoligonucleotide. Preferably, the detection step uses a homogeneousdetection method such as described in detail previously in Arnold et al.Clinical Chemistry 35:1588-1594 (1989), and U.S. Pat. Nos. 5,658,737(Nelson et al.), and 5,118,801 and 5,312,728 (Lizardi et al.). Themethods of the present invention may also include an optional step ofpurifying the target HIV-1 from other sample components beforeamplification, using any of a variety of known purification methods.Methods of purifying nucleic acids are well known in the art (Sambrooket al., Molecular Cloning, A Laboratory Manual, 2^(nd) ed. (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) at §§1.23-1.40, 2.73-2.80, 4.26-4.32 and 7.3-7.35). A preferred embodimentuses a purification step that is relatively fast, involving a minimum ofsteps, such as a solution phase hybridization disclosed in detail in PCTNo. WO 9850583 (Weisburg et al.). Briefly, the purification usesoligonucleotides that hybridize to the target and to immobilizedoligonucleotides on a removable solid support, such as magneticparticles (Whitehead et al., U.S. Pat. Nos. 4,554,088 and 4,695,392), toseparate the target nucleic acid from other sample components.

[0025] The present invention provides methods for detecting HIV-1nucleic acids present in human biological samples, such as tissue orbody fluid samples. A “biological sample” includes any tissue, bodyfluid or material derived from a living or dead human which may containHIV-1 nucleic acid, including, for example, peripheral blood, plasma,serum, lymph, bone marrow, cervical swab samples, lymph node tissue,respiratory tissue or exudates, gastrointestinal tissue, urine, feces,semen or other body fluids, tissues or materials. Using standardmethods, the biological sample may be treated to physically ormechanically disrupt tissue or cell structure, to release intracellularcomponents into an aqueous or organic solution to prepare nucleic acidsfor further analysis.

[0026] “Nucleic acid” refers to a multimeric compound comprisingnucleosides or nucleoside analogs which have nitrogenous heterocyclicbases, or base analogs, which are linked by phosphodiester bonds to forma polynucleotide. Nucleic acids include conventional RNA and DNA andanalogs thereof. The “backbone” of a nucleic acid may be made up of avariety of known linkages, including one or more of sugar-phosphodiesterlinkages, peptide-nucleic acid bonds (found in “peptide nucleic acids”or “PNA” as described in U.S. Pat. No. 5,539,082 (Nielsen et al.)),phosphorothioate linkages, methylphosphonate linkages or combinationsthereof. Sugar moieties may be either ribose or deoxyribose, or knownsubstitutions of such sugars, such as 2′ methoxy and 2′ halidesubstitutions (e.g., 2′-F). Nitrogenous bases may be conventional bases(A, G, C, T, U), analogs thereof, e.g., inosine (“I”) or nebularine(“N”) or synthetic analogs (The Biochemistry of the Nucleic Acids 5-36,Adams et al., ed., 11^(th) ed., 1992; Lin & Brown, 1989, Nucl. AcidsRes. 17:10373-83; Lin & Brown, 1992, Nucl. Acids Res. 20: 5149-52)),derivatives of purine or pyrimidine bases (e.g., N⁴-methyldeoxygaunosine, deaza- or aza-purines and deaza- or aza-pyrimidines,pyrimidine bases having substituent groups at the 5 or 6 position,purine bases having a substituent at the 2, 6 or 8 positions,2-amino-6-methylaminopurine, O⁶-methylguanine, 4-thio-pyrimidines,4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, andO⁴-alkyl-pyrimidines; see, PCT No. WO 9313121 (Cook)) and “abasic”residues where the polymer backbone includes no nitrogenous base at thatposition (U.S. Pat. No. 5,585,481 ( Arnold et al.)). A nucleic acid maycomprise only conventional sugars, bases and linkages, as found innaturally occurring RNA or DNA, or may include a combination ofconventional components and substitutions (e.g., conventional baseslinked via a 2′ methoxy backbone, or a polymer including conventionalbases and one or more base analogs).

[0027] The backbone composition of an oligomer may affect the stabilityof a hybridization complex formed between an oligomer and acomplementary nucleic acid strand. Embodiments of the oligomers of thepresent invention may include peptide linkages as in PNA,sugar-phosphodiester linkages, 2′ methoxy linkages in part or all of theoligomer, or derivatives thereof. An altered oligomer backbone, relativeto standard DNA or RNA, may enhance hybridization complex stability. Forexample, an oligomer that is a PNA or having 2′-methoxy linkages(containing a 2′-O-methylribofuranosyl moiety; PCT No. WO 98/02582) or2′-F substituted RNA groups forms a stable hybridization complex withcomplementary 2′ OH RNA. The linkage joining two sugar groups may affecthybridization complex stability by affecting the overall charge or thecharge density, or steric interactions. Embodiments of oligomers mayinclude linkages with charged (e.g., phosphorothioates) or neutral(e.g., methylphosphonates) groups to affect complex stability.

[0028] The present invention includes amplification oligonucleotides oroligomers to specifically amplify HIV-1 target sequences and probeoligonucleotides or oligomers to detect the HIV-1 target sequences ortheir amplification products. “Oligonucleotide” and “oligomer” refer toa polymeric nucleic acid having generally less than 1,000 residues,including those in a size range having a lower limit of about 2 to 5residues and an upper limit of about 500 to 900 residues. In preferredembodiments, oligomers are in a size range having a lower limit of about5 to about 15 residues and an upper limit of about 100 to 200 residues.More preferably, oligomers of the present invention are in a size rangehaving a lower limit of about 10 to about 15 residues and an upper limitof about 17 to 100 residues. Although oligomers may be purified fromnaturally occurring nucleic acids, they are generally synthesized usingany of a variety of well known enzymatic or chemical methods.

[0029] An “amplification oligonucleotide” or “amplification oligomer” isan oligonucleotide that hybridizes to a target nucleic acid, or itscomplement, and participates in a nucleic acid amplification reaction.Amplification oligomers include primers and promoter-primers in whichthe oligomer's 3′ end is extended enzymatically using another nucleicacid strand as the template. In some embodiments, an amplificationoligonucleotide contains at least about 10 contiguous bases, and morepreferably about 12 contiguous bases, that are complementary to a regionof the target sequence (or its complementary strand), and optionally maycontain other bases that do not bind to the target sequence or itscomplement. For example, a promoter-primer may contain target-bindingbases and additional 5′ bases that include a promoter sequence that doesnot hybridize to the target sequence. Contiguous target-binding basesare preferably at least about 80%, and more preferably about 90% to 100%complementary to the sequence to which it binds. An amplificationoligomer is preferably about 10 to about 60 bases long and may includemodified nucleotides or base analogs.

[0030] Embodiments of the present invention use amplification oligomersto specifically amplify regions of the HIV-1 genome, specificallyregions the gag and pol genetic sequences. These amplificationoligonucleotides include the sequences of SEQ ID NO:5 to SEQ ID NO:22and SEQ ID NO:33 to SEQ ID NO:68. Some amplification oligomers that arepromoter-primers include promoter sequences (SEQ ID NO:5 to SEQ ID NO:10and SEQ ID NO:33 to SEQ ID NO:45). Preferred T7 promoter sequencesincluded in promoter-primers are shown in SEQ ID NO:1 to SEQ ID NO:4.Those skilled in the art will appreciate that an oligomer that canfunction as a primer (i.e., one that hybridizes specifically to a targetsequence and has a 3′ polymerase-extendable end) can be modified toinclude a 5′ promoter sequence, and thus become a promoter-primer.Similarly, any promoter-primer sequence can function as a primerindependent of its promoter sequence (such as the sequences shownwithout promoters in SEQ ID NO:11 to SEQ ID NO:16 and SEQ ID NO:46 toSEQ ID NO:58).

[0031] By “amplify” or “amplifiication” is meant a procedure to producemultiple copies of a target nucleic acid sequence or its complement orfragments thereof (i.e., the amplified product may contain less than thecomplete target sequence). For example, fragments may be produced byamplifying a portion of the target nucleic acid by using anamplification oligonucleotide which hybridizes to, and initiatespolymerization from, an internal position of the target nucleic acid.Known amplification methods include, for example, replicase-mediatedamplification, polymerase chain reaction (PCR) amplification, ligasechain reaction (LCR) amplification, strand-displacement amplification(SDA) and transcription-associated or transcription-mediatedamplification (TMA). Replicase-mediated amplification uses QB-replicaseto amplify RNA sequences (U.S. Pat. No. 4,786,600 (Kramer et al.); PCTNo. WO 9014439). PCR amplification uses DNA polymerase, primers foropposite strands and thermal cycling to synthesize multiple copies ofDNA or cDNA (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159 (Mulliset al.); Mullis et al.,1987, Methods in Enzymology 155: 335-350). LCRamplification uses at least four different oligonucleotides to amplifycomplementary strands of a target by using cycles of hybridization,ligation, and denaturation (EP No. 0 320 308). SDA uses a primer thatcontains a recognition site for a restriction endonuclease and anendonuclease that nicks one strand of a hemimodified DNA duplex thatincludes the target sequence, followed by a series of primer extensionand strand displacement steps (U.S. Pat. Nos. 5,422,252 and U.S. Pat.No. 5,470,723 (Walker et al.)). An isothermal strand-displacementamplification method that does not rely on endonuclease nicking is alsoknown (U.S. Pat. No. 6,087,133 (Dattagupta et al.)).Transcription-associated or transcription-mediated amplification uses aprimer that includes a promoter sequence and an RNA polymerase specificfor the promoter to produce multiple transcripts from a target sequence,thus amplifying the target sequence (U.S. Pat. Nos. 5,399,491,5,480,784, 5,824,518 and 5,888,779 (Kacian et al.), 5,437,990 (Burg etal.), 5,409,818 (Davey et al.), 5,554,516 and 5,766,849 (McDonough etal.), 5,130,238 (Malek et al.), 4,868,105 and 5,124,246 (Urdea et al.),and 5,786,183 (Ryder et al.)), PCT Nos. WO 8801302 and WO 8810315(Gingeras et al.)).

[0032] Preferred embodiments of the present invention amplify the HIV-1target sequences using the present amplification oligomers in atranscription-mediated amplification (TMA) reaction. One skilled in theart will appreciate that these amplification oligonucleotides canreadily be used in other methods of nucleic acid amplification that usespolymerase-mediated primer extension.

[0033] “Transcription-mediated amplification” refers to nucleic acidamplification that uses an RNA polymerase to produce multiple RNAtranscripts from a nucleic acid template. The amplification reactionemploys an RNA polymerase, a DNA polymerase, an RNase H activity,ribonucleoside triphosphates, deoxyribonucleoside triphosphates, and apromoter-primer, and may include one or more additional amplificationoligonucleotides. Preferred embodiments use the methods disclosed indetail in U.S. Pat. Nos. 5,399,491, 5,480,784, 5,824,518 and 5,888,779(Kacian et al.), 5,554,516 and 5,766,849 (McDonough et al.), and5,786,183 (Ryder et al.)).

[0034] Following amplification of the HIV-1 target sequences, theamplified products are detected using hybridization to probes that allowdetection of a hybridization complex formed between the amplifiedsequence and the probe oligonucleotide sequence. In some embodiments,the probe is labeled and the signal detected from the hybridizationcomplex is produced from the labeled probe. In other embodiments,amplified products are labeled and hybridized to a probe and thedetected signal is produced from the labeled product in thehybridization complex. In embodiments which provide sequence informationin the detection step, the amplified nucleic acid is hybridized to anarray of oligonucleotide probes (U.S. Pat. Nos. 5,837,832 and 5,861,242(Chee et al.)) and the detected signals are analyzed using acomputerized system (U.S. Pat. Nos. 5,733,729 and 6,066,454 (Lipshutz etal.)), to produce a nucleic acid sequence from “base calls” by thesystem.

[0035] A “probe” refers to a nucleic acid oligomer that hybridizesspecifically to a nucleic acid target sequence, under conditions thatpromote hybridization, thereby allowing detection of the targetsequence. Detection may either be direct (i.e., resulting from a probehybridizing directly to the target sequence) or indirect (i.e.,resulting from a probe hybridizing to an intermediate molecularstructure that links the probe and target sequences). The “targetsequence” of a probe refers to a sequence within a nucleic acid,preferably in an amplified nucleic acid, which hybridizes specificallyto at least a portion of a probe oligomer. A probe may comprisetarget-specific sequences and other sequences that contribute to aprobe's three-dimensional conformation (see U.S. Pat. Nos. 5,118,801 and5,312,728 (Lizardi et al.)). Sequences that are “sufficientlycomplementary” allow stable hybridization of a probe oligomer to itstarget sequence under hybridization conditions, even if the probe andtarget sequences are not completely complementary by standard basepairing (G:C, A:T or A:U pairing). “Sufficiently complementary” probesequences may contain one or more residues (including abasic residues)that are not 100% complementary, but which, due to the probe's entirebase sequence are capable of specifically hybridizing with anothersequence in the hybridization conditions. Appropriate hybridizationconditions are well known in the art, can be predicted readily based onbase sequence composition, or can be determined empirically by usingroutine testing (e.g., see Sambrook et al., Molecular Cloning, ALaboratory Manual, 2^(nd) ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989) at §§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and11.47-11.57, particularly §§ 9.50-9.51, 11.12-11.13, 11.45-11.47 and11.55-11.57). In preferred embodiments, probes include contiguous basesthat are at least about 80%, more preferably at least about 90%, andmost preferably about 100% complementary to the target sequence.

[0036] For detection, the probe may be labeled, i.e., joined directly orindirectly to a detectable molecular moiety or a compound that leads toa detectable signal. Direct labeling can occur through bonds orinteractions that link the label to the probe, including covalent bondsand non-covalent interactions (e.g. hydrogen bonding, hydrophobic andionic interactions), or formation of chelates or coordination complexes.Indirect labeling occurs through use of a bridging moiety (a “linker”),that joins a label to the probe, and which can amplify a detectablesignal (e.g., see PCT No. WO 95/16055 (Urdea et al.)). Labels are wellknown and include, for example, radionuclides, ligands (e.g., biotin,avidin), enzymes and/or enzyme substrates, reactive groups, redox activemoieties such as transition metals (e.g., Ru), chromophores (e.g., amoiety that imparts a detectable color), luminescent componds (e.g.,bioluminescent, phosphorescent or chemiluminescent labels) andfluorescent compounds. Those skilled in the art will appreciate that alabeled probe may be a mixture of labeled and unlabeled oligonucleotidesthat hybridize specifically to the target sequence, to optimize thespecific activity of the probe reagent for detection. In someembodiments, the label on a probe is detectable in a homogeneous assaysystem, i.e., in a mixture, bound labeled probe exhibits a detectablechange, such as stability or differential degradation, compared tounbound labeled probe. A preferred label for use in a homogenous assayis a chemiluminescent compound (U.S. Pat. Nos. 5,656,207 (Woodhead etal.), 5,658,737 (Nelson et al.), and 5,639,604 (Arnold, Jr., et al.)).Preferred chemiluminescent labels include acridinium ester (“AE”)compounds, which may be standard AE or derivatives thereof (e.g.,naphthyl-AE, ortho-AE, 1- or 3-methyl-AE, 2,7-dimethyl-AE,4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE,ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE,ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE, 1- or3-methyl-meta-difluoro-AE, and 2-methyl-AE). Methods of attaching labelsto nucleic acids and detecting labels are well known in the art (e.g.,see Sambrook et al., Molecular Cloning, A Laboratory Manual, 2^(nd) ed.(Cold Spring Harbor Laboratory Press, Cold Spring Habor, N.Y., 1989),Chapter 10, U.S. Pat. Nos. 5,658,737 (Nelson et al.), 5,656,207(Woodhead et al.), 5,547,842 (Hogan et al.), and 5,283,174 (Arnold, Jr.et al.), and PCT No. WO 9802582 (Becker)).

[0037] Methods of the present invention may optionally include a step ofpurifying the HIV-1 target nucleic acid from other sample componentsbefore amplification. By “purifying” is meant that the target isseparated from one or more components of the biological sample (e.g.,other nucleic acids, proteins, carbohydrates or lipids). Preferably, apurifying step removes at least about 70%, and more preferably about 90%or more of the other sample components. In embodiments that include sucha purification step, the HIV-1 target nucleic acid is hybridized to a“capture oligomer” that specifically joins the HIV-1 target sequence toan immobilized oligomer (i.e., attached to a solid support) based onnucleic acid hybridization, as previously described in detail (PCT No.WO 98/50583). This step is advantageous because it involves twosolution-phase hybridizations (hybridization of the capture oligomer andthe target, followed by hybridization of the target:capture oligomercomplex to the immobilized oligomer to produce a target:captureoligomer:immobilized oligomer complex) that permit rapid separation ofthe target from the other sample components.

[0038] By “consisting essentially of” is meant that additionalcomponent(s), composition(s) or method step(s) that do not materiallychange the basic and novel characteristics of the present invention maybe included in the compositions or methods of the present invention.Such characteristics include the ability to amplify and detect HIV-1sequences present in a sample.

[0039] Unless defined otherwise, all scientific and technical terms usedherein have the same meaning as commonly understood by those skilled inthe relevant art. General definitions of many of the terms used hereinare provided, for example, in Dictionary of Microbiology and MolecularBiology, 2nd ed. (Singleton et al., 1994, John Wiley & Sons, New York,N.Y.). Unless otherwise described, the techniques employed orcontemplated herein are standard methods well known to one of ordinaryskill in the art. The examples illustrate some of the preferredembodiments.

[0040] The present invention includes nucleic acid oligomers and methodsfor detecting HIV-1 nucleic acid present in a human biological sample.To determine appropriate DNA sequences for use as amplificationoligomers, known HIV-1 sequences, generally of known subtypes andincluding partial or complementary sequences available from publiclyaccessible databases (e.g., GenBank), were aligned by matching regionsof the same or similar sequences and compared using well known molecularbiology techniques. Although use of algorithms may facilitate sequencecomparisons, those skilled in the art can readily perform suchcomparisons without the aid of an algorithm. Amplification oligomerswere designed that would amplify portions of the HIV-1 gag and pol genes(based on the sequence of HIV-1 HXB2, GenBank Acc. No. KO 3455) providedthat: the oligomer sequences do not contain known drug-resistancemutations, the oligomer combinations each amplify about 300 to 600 nt,the oligomers contain a minimum of predicted secondary structure basedon well-known methods for predicting nucleic acid structure, thesequences contain few known polymorphic bases (intra- or inter-subtypepolymorphisms) and, for residues in which polymorphisms occur, that thebase found in the majority of compared sequences for that position or abase analog (e.g., nebularine) is used at that position. Sequencecomparisons used in designing amplification oligomers, generally were:for gag region, 22 subtype A, 41 subtype B, 12 subtype C, 11 subtype D,6 subtype F, 4 subtype G, 3 subtype H, and 2 subtype J sequences; forthe Protease region, 22-34 subtype A, 36-41 subtype B, 15 subtype D, 26subtype F, and 8 subtype G sequences; and for the RT region, 32-44subtype A, 38-46 subtype B, 24 subtype C, 9-15 subtype D, 5-26 subtypeF, and 8 subtype G sequences.

[0041] Probe oligomers were similarly designed, selecting for sequencescomplementary to those that occur within the amplified sequences. DNAoligomers were synthesized using well known synthetic methods and testedfor their efficiency as amplification or probe oligomers. Labeled probeswere labeled with an AE compound attached via a linker, substantially asdescribed in detail in U.S. Pat. No. 5,639,604 (see column 10, line 6 tocolumn 11, line 3, and Example 8). The efficiency of oligomers wastested in the amplification and detection systems described herein, andin some cases, oligomer sequences were modified to optimizeamplification or detection, e.g., by changing one or more residues,substituting a base analog, or by modifying part or all of the oligomerbackbone (e.g., substituting 2′-O-methyl RNA for DNA).

[0042] Based on these analyses, the amplification oligonucleotidesdescribed herein were identified. Amplification oligonucleotides mayoptionally include a promoter sequence for producing transcripts fromamplified target sequences, and preferred T7 promoter sequences areshown in SEQ ID NO:1 to SEQ ID NO:4. For amplification oligomers thatinclude a T7 promoter sequence, the primer sequences have been shownwith the T7 promoter sequence (SEQ ID NO:5 to SEQ ID NO:10 and SEQ IDNO:33 to SEQ ID NO:45) and without a T7 promoter sequence (SEQ ID NO:11to SEQ ID NO:16 and SEQ ID NO:46 to SEQ ID NO:58). Those skilled in theart will appreciate that a amplification oligomer specific for HIV-1,with or without a promoter sequence, may be useful as a primer underappropriate amplification conditions.

[0043] Referring to FIGS. 1 and 2, the upper portion illustrates the gagand pol regions of the HIV-1 genome (shaded overlapping regions labeledgag and pol and the lower portion shows the approximate sizes andlocations of the regions (double lines labeled underneath as Gag1, Gag2,Prt, RT1, RT3 and RT4) amplified using the amplification primers. Theseamplified regions include the characteristics: Gag1 contains thecleavage site between Gag P24 and Gag P17; Gag2 contains other Gagcleavage sites; Prt contains two cleavage sites at the 3′ end of Gag,the protease region and the cleavage site between protease and reversetranscriptase (RT); RT1 contains codons between 41 and 190 of RT; RT3contains codons between 200 and 350 of RT; and RT4 contains codons atthe 3′ end of Rt and the cleavage site between RT and RNase H. FIGS. 1and 2 illustrate the relative sizes and locations of the amplifiedregions, although the absolute sizes of amplified regions may varyslightly depending on the particular combination of amplificationoligomers used. For example, Gag sequences amplified using primershaving the sequences of SEQ ID NO:5 and SEQ ID NO:17 produce a 260 ntamplification product (Gag1), and SEQ ID NO:6 and SEQ ID NO:18 produce a415 nt amplification product (Gag2). The protease (Prt) region amplifiedusing the primers having the sequences of SEQ ID NO:7 and SEQ ID NO:19produce a 574 nt amplification product. Three reverse transcriptase (RT)sequences of the pol gene result from amplification using primers havingthe sequences of: SEQ ID NO:8 and SEQ ID NO:20 to produce a 532 ntproduct (RT1), SEQ ID NO:9 and SEQ ID NO:21 to produce a 464 nt product(RT3), and SEQ ID NO:10 and SEQ ID NO:22 to produce a 384 nt product(RT4). Other combinations of related amplification oligomer sequencescan similarly be used to amplify these regions, for example: for Gag1(SEQ ID NO:33 and SEQ ID NO:17 or SEQ ID NO:59), for Gag2 (SEQ ID NO:34or SEQ ID NO:36 and SEQ ID NO:60), for Prt (SEQ ID NO:37 or SEQ ID NO:38and SEQ ID NO:19, SEQ ID NO:61 or SEQ ID NO:62), for RT1 (SEQ ID NO:39,SEQ ID NO:40 or SEQ ID NO:41 and SEQ ID NO:20, SEQ ID NO:63, SEQ IDNO:64 or SEQ ID NO:65), for RT3 (SEQ ID NO:42 or SEQ ID NO:43 and SEQ IDNO:66), and for RT4 (SEQ ID NO:44 or SEQ ID NO:45 and SEQ ID NO:67 orSEQ ID NO:68). In preferred embodiments, the amplification oligomers ofSEQ ID NO:18 to SEQ ID NO:22, and SEQ ID NO:35 include 2′-O-methoxylinkages for the backbone of one or more 5′ residues, preferably forresidues 1 to 4.

[0044] For detection of the amplified target sequences, probe sequencescomplementary to the amplified sequences were designed, and preferredembodiments of such probes have the sequences of SEQ ID NO:23 to SEQ IDNO:29, SEQ ID NO:69 and SEQ ID NO:70. More specifically, a probe of SEQID NO:23 hybridizes to Gag1 amplification products or amplicons, a probeof SEQ ID NO:24 hybridizes to Gag1 amplicons produced from an HIV-1subtype A template, probes of SEQ ID NO:25 and SEQ ID NO:69 hybridize toGag2 amplicons, a probe of SEQ ID NO:26 hybridizes to Prt amplicons,probes of SEQ ID NO:27 and SEQ ID NO:70 hybridize to RT1 amplicons, aprobe of SEQ ID NO:28 hybridizes to RT3 amplicons, and a probe of SEQ IDNO:29 hybridizes to RT4 amplicons. Any backbone may be used to link thebase sequence of a probe oligomer, and some embodiments include one ormore 2′-O-methoxy linkages, e.g., for SEQ ID NO:24 to SEQ ID NO:29, SEQID NO:69 and SEQ ID NO:70. When used as labeled probes, the probeoligomers may include any known detectable label or label that leads toproduction of a detectable signal. In some embodiments, the probeoligomers are labeled with an acridinium ester (AE) compound via alinker as described previously (U.S. Pat. No. 5,639,604 (Arnold, Jr. etal.)). For example, some embodiments of the probes were labeled asfollows: SEQ ID NO:23 between residues 9 and 10, SEQ ID NO:24 betweenresidues 10 and 11, SEQ ID NO:25 between residues 17 and 18, SEQ IDNO:26 between residues 9 and 10, SEQ ID NO:27 between residues 16 and17, SEQ ID NO:28 between residues 9 and 10, SEQ ID NO:29 betweenresidues 10 and 11, SEQ ID NO:69 between residues 11 and 12, and SEQ IDNO:70 between residues 11 and 12.

[0045] For those embodiments that use a capture oligomer to purify theHIV-1 target from other sample components, the capture oligomersequences were a combination of SEQ ID NO:30, SEQ ID NO:31 and SEQ IDNO:32. These capture probes include a 5′ portion of the sequence thatspecifically hybridizes with an HIV-1 sequence (pol or LTR sequences)and a 3′ poly-A “tail” portion that hybridizes with a complementaryimmobilized oligomer (dT₁₄) on a solid support (e.g., paramagneticparticles). Those skilled in the art will appreciate that any 3′ portionthat binds to a complementary immobilized sequence may be used in placeof the poly-dA tail and any backbone that permits hybridization may beused to link the base sequence of a capture oligomer.

[0046] Using these components, an assay to detect HIV-1 sequences in abiological sample includes the steps of optionally purifying the targetHIV-1 nucleic acid from a sample using one or more capture oligomers,amplifying the target HIV-1 region using at least two primers,preferably by using a transcription-mediated amplification reaction, anddetecting the amplified nucleic acid by hybridizing the amplifiednucleic acid with one or more probes that hybridize specifically to theamplified nucleic acid. If the detection step uses a labeled probe, thenthe a signal resulting from the bound labeled probe is detected. If thedetection step uses hybridization to an array of probes attached to asolid support (a “DNA chip”), then the amplification product may belabeled before it is hybridized to the DNA chip for detection of probeloci that specifically bind to the amplified product. Amplicons may belabeled as part of the amplification step or after amplification. Apreferred method for labeling the amplicons after amplification alsofragments the product into smaller portions for detection on a DNA chip(see PCT No. WO 00/65926 and PCT No. PCT/IB99/02073 for details).

[0047] If an optional purification step before amplification is used, itis preferably performed in a single reaction vessel using a minimum ofhandling steps, such as by using the two-step hybridization proceduredescribed in PCT No. WO 98/50583. Briefly, a capture oligomer is addedto a sample containing HIV-1 target nucleic acid under a firsthybridizing condition in which one portion of the capture oligomerspecifically hybridizes to the HIV-1 target sequence, producing acapture oligomer:HIV-1 RNA complex. Then, under a second hybridizingcondition, a second portion of the capture probe hybridizes to acomplementary oligomer sequence immobilized on a solid support such as amagnetic bead, producing an immobilized oligomer:capture oligomer:HIV-1RNA complex. For both hybridization conditions, the sample is in amixture of salts, detergent and buffer (e.g., 400 mM HEPES, 1-10%lithium lauryl sulfate (LLS), 240-832 mM LiOH, 0-783 mM LiCl, pH7.6-8.0), the capture oligomer(s), and immobilized oligomers on solidparticles, and the two different hybridization conditions are obtainedby incubating at a first and a second temperature. Then the solidsupport with the attached immobilized oligomer:capture oligomer:HIV-1RNA complex is physically separated from other sample components topurify the target HIV-1 RNA. The solid support with the attached nucleicacid complex can be washed using standard methods (e.g., rinsing with abuffered aqueous solution under conditions that allow the nucleic acidcomplex to remain hybridized), to further purify the HIV-1 RNA. Ifmagnetic particles are the solid support, physical separation canreadily be accomplished by application of a magnetic field to thevessel. Following purification, the HIV-1 RNA-containing complexattached to the support may be used directly in amplification.

[0048] Amplifying the HIV-1 target using two or more amplificationoligomers can be accomplished using a variety of known nucleic acidamplification reactions that rely on primer extension to producemultiple copies of the target sequence or its complement. One embodimentuses transcription-mediated amplification substantially as described indetail previously (U.S. Pat. Nos. 5,399,491, 5,480,784, 5,554,516,5,766,849, 5,786,183, 5,824,518, and 5,888,779). Briefly,transcription-mediated amplification uses two primers (one being apromoter-primer that contains a promoter sequence for an RNApolymerase), a DNA polymerase (a reverse transcriptase), an RNApolymerase, and nucleosides (deoxyribonucleoside triphosphates,ribonucleoside triphosphates) with appropriate salts and buffers insolution to produce multiple RNA transcripts from a nucleic acidtemplate. Initially, a promoter-primer (also referred to as a P1 primer)hybridizes specifically to the target sequence and the reversetranscriptase creates a first strand cDNA by extension of the 3′ end ofthe promoter-primer, and its RNase H activity digests the RNA targetstrand in the RNA:cDNA complex. The first strand cDNA hybridizes withthe second primer (also referred to as a P2 primer) at a location 3′ ofthe promoter-primer sequence and the reverse transcriptase creates a newDNA copy by extension of the 3′ end of the P2 primer, thereby creating adouble-stranded DNA having a functional promoter sequence at one end.RNA polymerase binds to the functional promoter sequence and transcribesit to produce multiple transcripts (which are amplification products oramplicons). Each transcript can serve as a template for another cycle ofreplication, i.e., the P2 primer binds to the amplicon, and reversetranscriptase creates a cDNA to which the P1 promoter binds and reversetranscriptase make a double-stranded DNA with a functional promoter atone end which binds the RNA polymerase to produce more transcripts.Thus, under substantially isothermal conditions, transcription-mediatedamplification produces many amplicons, i.e., about 100 to about 3,000RNA transcripts from a single template. Although embodiments describedin the examples use P1 promoter-primers that include a promoter sequencerecognized by T7 RNA polymerase, those skilled in the art willappreciate that any combination of a promoter sequence and itscorresponding RNA polymerase may be used (e.g., T3 polymerase).

[0049] Amplification reaction mixtures for transcription-mediatedamplification generally contain 1-2.5 mM Mg+⁺², 18-40 mM Tris (pH 8),0.5-3.5 mM ATP, 1.75-7.0 mM UTP, 5-16 mM GTP, 1.75-5.6 mM CTP, 0.75 mMdNTP, 25-37.5 mM KCl, optimized amounts of each primer, 2600-3000 Ureverse transcriptase and 2600-3600 U T7 RNA polymerase. A typicalreaction mixture includes 1 mM Mg⁺², 32.5 mM Tris (pH 8), 0.5-1.0 mMATP, 5.0 mM UTP, 5-9 mM GTP, 5 mM CTP, 0.75 mM dNTP, 37.5 mM KCl,primers, and 3000 U each of reverse transcriptase and T7 RNA polymerase.

[0050] Detecting the amplification products may use any step thatdetects specific hybridization of amplicon to one or more probesequences. If a labeled probe hybridizes to the amplicons, the label ispreferably one that can be detected in a homogeneous system (i.e., onethat does not require unbound probe to be separated from theamplicon:probe complexes for detection of bound probes). In someembodiments, the label is an AE compound from which produces achemiluminescent signal that is detected, as described in detailpreviously (U.S. Pat. Nos. 5,283,174, 5,656,744 and 5,658,737).Alternatively, amplicons or fragments thereof may be hybridized to anarray of probes as on a DNA chip and those probes that specificallyhybridize to the amplicons are detected to provide sequence informationabout the HIV-1 target from which the amplicons were produced. Thoseskilled in the art will appreciate that more than one procedure may beused to detect the amplification products produced from a singleamplification reaction. For example, a portion of the amplificationreaction may be subjected to a labeled probe hybridization procedure toprovide a positive or negative response, indicating the presence orabsence of HIV-1 specific amplification products in the reaction,thereby indicating that the sample was positive or negative for HIV-1.For reactions that test positive, another portion of the amplificationreaction may be further assayed using hybridization to a DNA probe arrayto provide sequence information on the HIV-1 present in the sample,thereby providing further more detailed diagnostic information.

[0051] Although any one of the gag and pol regions amplified by usingthe disclosed amplification oligomers may be detected to supplydiagnostic information about the sample, amplification and detection ofmore than one region potentially provides more diagnostic information,for example, by detecting multiple genetic markers associated withdrug-resistance. Individual gag and pol regions may be amplifiedseparately and the amplicons then pooled for detection, but to simplifythe assay multiple regions were amplified in multiplex reactions. Thatis, each multiplex reaction includes multiple amplification oligomers toamplify multiple HIV-1 target regions in a single reaction vessel.

[0052] Multiplex reactions were designed to amplify multiple targetsequences while avoiding potential interference between amplificationoligomers for hybridization to their respective target sequences orproduction of relatively small amplicons. For example, referring to FIG.1, the target regions labeled RT1 and RT3 overlap, as do the RT3 and RT4target regions. Thus, a relatively small target region could beamplified if a multiplex reaction included amplification oligomers forthe overlapping targets, e.g., from use of the combination of SEQ IDNO:9 or SEQ ID NO:15 (P1 primers for RT3) and SEQ ID NO:22 (P2 primerfor RT4). Therefore, multiplex reactions were designed to include primercombinations that will not produce small amplicons which would notprovide much diagnostic information but would consume substrates duringamplification. Different combinations of amplification oligomers weretested in multiplex reactions, including (1) amplification oligomers toamplify the Gag1, RT1 and RT4 target regions in one vessel andamplification oligomers to amplify the Gag2, Prt and RT3 target regionsin another vessel, (2) amplification oligomers to amplify the Gag1, Gag2and RT3 target regions in one vessel and amplification oligomers toamplify the Prt, RT1 and RT4 target regions in another vessel(illustrated in FIG. 1), and (3) amplification oligomers to amplify theGag2, RT1 and RT4 target regions in one vessel and amplificationoligomers to amplify the Gag1, Prt and RT3 target regions in anothervessel (illustrated in FIG. 2). Each of the reactions of these multiplexcombinations amplifies over 1 kb of HIV-1 target nucleic acid and thecombined two multiplex reactions for each combination cumulativelyamplify over 2.5 kb of HIV-1 sequence.

[0053] Some embodiments of the present invention are illustrated by theexamples that follow. In these examples, the HIV-1 RNA used in nucleicacid amplification was from virus present in supernatants of lymphocytecultures infected with virus subtypes A, B or C, purified HIV-1 RNAisolated from culture supernatants, transcripts produced from clonedHIV-1 sequences, or plasmas obtained from patients infected with HIV-1,where the viral subtype was determined by sequencing or by antigenspresent in the viral envelop of viruses in the plasma.

Example 1 Amplification and Detection of a Gag Target Sequence fromDifferent Subtypes of HIV-1

[0054] Samples were prepared containing known amounts of HIV-1 RNA fromthree different subtypes (A, B and C), which were then amplified usingprimers specific for the Gag1 target region in a transcription-mediatedamplification reaction. Following amplification, the amplificationproducts were detected by using two different labeled probes for theGag1 amplicons and a mixture of the two different labeled probes.

[0055] In the first test HIV-1 subtype B RNA was mixed with water toachieve 500 copies per reaction tube, and HIV-1 subtype A viralsupernatant was mixed with a buffer (400 mM HEPES, pH 7.6, 10% (w/v)lithium lauryl sulfate (LLS), 350 mM LiOH, 783 mM LiCl) to achieve 500copies per reaction tube. For amplification, each reaction contained thesample and 7.5 pmol each of the amplification oligomers having SEQ IDNO:5 (a T7 primer-promoter or P1 primer) and SEQ ID NO:17 (a P2 primer)in a reaction mixture (40 mM Tris, pH 7.5, 17.5 mM KCl, 20 mM MgCl₂, 5%polyvinylpyrrolidone, 1 mM each dNTP, 4 mM each rNTP). The reactionmixtures were covered with a layer (200 μl) of inert oil to preventevaporation, and incubated at 60° C. for 10-15 min, and then at 41.5-42°C. for 5 min. For each reaction mixture, about 3000 U each of reversetranscriptase and T7 RNA polymerase were added, mixed, and the targetHIV-1 nucleic acid was amplified at 41.5-42° C. for 2 hr.

[0056] Following amplification, detection of the amplified Gag targetregion was detected by using AE-labeled probes having SEQ ID NO:23, SEQID NO:24 or a mixture of these probes (0.05-0.1 pmol each), andchemiluminescence was detected from bound probes using previouslydescribed methods (U.S. Pat. No. 5,658,737 at column 25, lines 27-46;Nelson et al., 1996, Biochem. 35:8429-8438 at 8432). The detectedchemiluminescence was expressed in relative light units (RLU). Negativecontrols were similarly treated but contained no HIV-1 target nucleicacid.

[0057] Table 1 presents the detected RLU results for each probe (mean oftriplicate assays, except one negative control assay for the “ProbeMixture”). These results show that both subtypes of HIV-1 were amplifiedusing the amplification oligomers for the Gag1 target region but theprobe of SEQ ID NO:23 only detected amplified Gag1 products from subtypeB, whereas the probe of SEQ ID NO:24 detected amplified products fromboth subtypes A and B. The results obtained using the probe mixtureappear to be additive of the results obtained with the individual probesfor the two subtypes. TABLE 1 Amplification and Detection (RLU) of HIV-1Subtype A and Subtype B Gag1 Target Region SEQ ID NO: HIV-1 Subtype 23Probe SEQ ID NO:24 Probe Probe Mixture A 1.12 × 10⁴ 5.55 × 10⁶ 5.67 ×10⁶ B 5.13 × 10⁶ 4.55 × 10⁶ 9.04 × 10⁶ Negative Control 1.31 × 10⁴ Notdetermined 2.25 × 10⁴

[0058] In another test, similar assays were performed using HIV-1subtypes A, B and C at 250, 50 or 25 copies of virus RNA per reaction,and using the probe mixture (SEQ ID NO:23 and SEQ ID NO:24). Forsubtypes B and C, HIV-1 RNA was the target, and, for subtype A, viralsupernatant contained the target nucleic acid. Table 2 presents theresults of this test (mean RLU for 6 samples for each condition). Thenegative controls, i.e., no HIV-1 target present, produced 1.37×10⁴ RLU(mean of duplicate assays). TABLE 2 Detection of Amplified Gag1 TargetRegion By Using a Labeled Probe Mixture HIV-1 250 Copies Subtype ofTarget 50 Copies of Target 25 Copies of Target A 4.73 × 10⁶ 2.80 × 10⁶8.76 × 10⁵ B 2.79 × 10⁶ 1.77 × 10⁶ 1.52 × 10⁶ C 4.51 × 10⁶ 4.46 × 10⁶1.78 × 10⁶

[0059] These results show that the amplification oligonucleotidesamplified the Gag1 target region for HIV-1 subtypes A, B and C and theprobes detected the amplified sequences. The assay system had asensitivity of at least 50 copies of target for all of the subtypes. At25 copies of target per reaction, all of the subtypes were amplified anddetected (producing at least 10-fold more RLU than the negativecontrols) in at least half of the reactions (data not shown). Theseresults show that HIV-1 can be readily detected in a biological sampleusing the methods of the present invention.

[0060] Similar experiments were performed using the combinations ofamplification oligomers for each of the other individual targetsequences amplified (Gag2, Prt, RT1, RT3 and RT4). Probe oligomers werealso hybridized specifically to each of their respective targets insimilar homogeneous detection assays to produce detectable luminescentsignals proportional to the amount of amplified product produced in theamplification reaction.

Example 2 Sensitivity of Detection of HIV-1 Target Sequences of SubtypesA, B and C

[0061] This example shows that the amplification oligomers caneffectively amplify target sequences from the three subtypes of HIV-1that are most prevalent throughout the world, subtypes A, B, and C. Inthese experiments, HIV-1 target RNA was used in transcription-mediatedamplification reactions at 25, 50, 100, 250, 500 or 1000 copies perreaction, obtained from viral RNA, RNA transcripts from cloned HIV-1sequences or virus present in plasma. The individual target regions(Gag1, Gag2, Prt, RT1, RT3 and RT4) were amplified in separate reactionsand detected by hybridization with AE-labeled probes using substantiallythe conditions described in Example 1. The results (RLU for eachsubtype) of some of these experiments are shown in Table 3 (reportingaverage RLU obtained from at least triplicate reactions, except for thePrt results reported for plasma virus detection which were results fromone test). The sensitivity is shown by the number of copies of thetarget (for the different sources of virus (“V”), transcript (“T”) orinfected plasma (“P”)) present in the sample which were amplified. TABLE3 Sensitivity of Detection of Amplified HIV-1 Target Regions UsingAE-labeled Probes Subtype A Subtype B Subtype C Target Primers (copies &source) (copies & source) (copies & source) Gag1 SEQ ID NO:5 7.1 × 10⁶(100 T) 4.3 × 10⁶ (50 T) SEQ ID NO:17 2.8 × 10⁶ (50 V) 1.5 × 10⁶ (25V)1.8 × 10⁶ (25 V) Prt SEQ ID NO:7 2.2 × 10⁶ (500 T) 3 × 10⁶ (250 T) 1.7 ×10⁶ (250 T) SEQ ID NO:19 5.9 x 10⁶ (100 V) 3 × 10⁶ (100 P) >3 × 10⁶ (500P) >3.5 × 10⁶ (100 P) RT4 SEQ ID NO:10 2.1 × 10⁶ (50 T) 3.8 × 10⁶ (50 T)5.7 × 10⁶ (50 T) SEQ ID NO:22 >2.5 × 10⁶ (25 V) >2.2 × 10⁶ (25 V) >3.6 ×10⁶ (25 V)

[0062] These results show that for all three subtypes, the sensitivityof detection was at least 100 copies of target, and often was less than100 copies (e.g., 25-50 copies of virus for Gag1 for all threesubtypes). Similar results were obtained for the other HIV-1 targetregions (data not shown), all having a sensitivity of detection of atleast 200 copies of target for the three subtypes in most of the assaysperformed using the amplification oligomers and detection probes of thisinvention.

Example 3 Detection of HIV-1 Target Sequences in Multiplex Reactions

[0063] In this experiment, two separate multiplex reactions, eachamplifying three HIV-1 target regions were used to show that theindividual target regions can be amplified to produce detectableamplicons. Each amplification tube contained 200 copies of HIV-1 subtypeB RNA and amplification reagents substantially as described inExample 1. Amplification tube 1 contained primers to amplify the Gag2,RT1 and RT4 regions (SEQ ID NO:6 and SEQ ID NO:18, SEQ ID NO:8 and SEQID NO:20, including a 3′ blocked oligomer of SEQ ID NO:8, and SEQ IDNO:10 and SEQ ID NO:22). Amplification tube 2 contained primers toamplify the Gag1, Prt and RT3 regions (SEQ ID NO:5 and SEQ ID NO:17, SEQID NO:7 and SEQ ID NO:19, and SEQ ID NO:9 and SEQ ID NO:21). The amountsper reaction of each of these primers were as follows: in tube 1, 4 pmoleach of SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:18 and SEQ ID NO:22, 2.4pmol of SEQ ID NO:8, 12 pmol of SEQ ID NO:20 and 9.6 pmol of 3′ blockedSEQ ID NO:8; and in tube 2, 1 pmol of SEQ ID NO:5, 4 pmol of SEQ IDNO:17, 4.5 pmol of SEQ ID NO:7, 5 pmol each of SEQ ID NO:9 and SEQ IDNO:19, and 9.85 pmol of SEQ ID NO:21. For each set of reactions, fiveindividual tubes were prepared and amplified. Amplification reactionswere performed substantially as described in Example 1, and then half ofthe reaction (i.e., the equivalent of amplicons produced from 100 copiesof target HIV-1 RNA) was used in the detection step.

[0064] Detection was performed using AE labeled probes using thehomogeneous detection assay substantially as described previously (U.S.Pat. No. 5,658,737 at column 25, lines 27-46; Nelson et al., 1996,Biochem. 35:8429-8438 at 8432) and chemiluminescence was detected as RLUfor each amplification reaction and for each probe using a calibratedluminometer (LEADER® HC450, Gen-Probe Incorporated, San Diego, Calif.).The labeled probes used (0.1 pmol/reaction) were: SEQ ID NO:24 to detectGag1, SEQ ID NO:69 to detect Gag2, SEQ ID NO:26 to detect Prt, SEQ IDNO:70 to detect RT1, SEQ ID NO:28 to detect RT3 and SEQ ID NO:29 todetect RT4. To optimize the specific activities of the probes fordetection, the probe reagents generally were a mixture of labeled andunlabeled probes for each of the probe oligomers.

[0065] The results of these tests, reported as the mean of the fivereactions for each condition, are shown in Table 4 (negative controlsare not shown). These results show that all three of the targetsequences were amplified in each of the multiplex reactions to produceamplicons that were specifically detected by their corresponding probesspecific for the amplified sequences. TABLE 4 Multiplex AmplificationReaction Products Detected by Labeled Probe Hybridization (RLU) Tube 1Gag2 RTI RT4 2.5 × 10⁶ 1.4 × 10⁶ 6.3 × 10⁵ Tube 2 Gag1 Prot RT3 1.5 ×10⁵ 8.5 × 10⁵ 1.6 × 10⁶

[0066] In other experiments, different ratios of amplification oligomerswere used in the same multiplex combinations as tested above to providesimilar amplification results. For example, another multiplexcombination used the following amounts of primers per reaction tube 1:4pmol each of SEQ ID NO:6, SEQ ID NO:10, and SEQ ID NO:22, 3 pmol of SEQID NO:18, 2.4 pmol of SEQ ID NO:8, 9.6 pmol each of SEQ ID NO:20 and 3′blocked SEQ ID NO:8.

Example 4 Detection of HIV-1 Target Sequences from Clinical Samples

[0067] This example describes detection of HIV-1 target sequencespresent in biological samples taken from HIV-1 patients whose symptomsindicate that the infection has become drug resistant (viral reboundduring drug treatment). Multiplex amplification reactions were used toproduce amplified nucleic acid from multiple targets in a singlereaction tube for detection. The method detected amplified productshybridized to an array of DNA probes to provide amino acid informationat residues commonly associated with HIV-1 drug resistance in the Gag,Protease and RT regions.

[0068] Plasma was collected from three HIV-positive patients beingtreated with combinations of drugs who were experiencing increased viralload. Patients 1 and 2 were both being treated with one drug combination(d4T, 3TC and NELFINAVIR™) and had viral loads of 6,130 and 10,000copies/ml, respectively; Patent 3 was treated with another drugcombination (ABACAVIR™, EFAVIRENZ™ and NELFINAVIR™) and had a viral loadof 10,000 copies/ml. From each patient, 0.5 ml of plasma was dispensedinto two separate tubes and mixed with 0.5 ml of lysis buffer (0.4 MHEPES, pH 7.6, 0.35 M LiOH, 0.22 M LLS) and incubated at 60° C. for 20min. Released viral RNA was purified from other sample components byhybridization to three capture oligonucleotides added to the mixture(3.5 pmol/test of each of SEQ ID NO:30 and SEQ ID NO:32, and 10pmol/test of SEQ ID NO:31) and dT₄ oligonucleotides immobilized onmagnetic particles; the mixture was incubated at 55-60° C. for 15-20 minand then at 18-25° C. for 10-15 min. The magnetic particles werepelleted using a magnetic field and unbound sample components wereaspirated away; the magnetic particles with bound HIV-1 RNA were washedtwice (1.5 ml of washing buffer). The washed particles were suspended in75 μl of amplification buffer (50 mM HEPES, pH 7.5, MgCl₂, KCl,glycerol, dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, UTP and amplificationoligonucleotides). The amplification oligonucleotides for each targetregion were: SEQ ID NO:33 and SEQ ID NO:17 for Gag1; SEQ ID NO:34 andSEQ ID NO:60 for Gag2; SEQ ID NO:41 and SEQ ID NO:19 for Protease; SEQID NO:41 and SEQ ID NO:20 for RT1; SEQ ID NO:9 and SEQ ID NO:21 for RT3;and SEQ ID NO:10 and SEQ ID NO:22 for RT4. Amplification reaction tube 1contained primers for amplifying the Gag1, Protease and RT3 targets; andamplification reaction tube 2 contained primers for amplifying the Gag2,RT1 and RT4 targets. Amplification reaction mixtures were incubated at60° C. for 10 min and then about 3000 U each of reverse transcriptaseand T7 RNA polymerase were added (in 25 μl of 8 mM HEPES, pH 7.5,170 mMTris, 70 mM KCl, 0.01% Phenol Red, 10% TRITON® X-100, 0.08% TRITON®X-102, 20% glycerol, 0.04 mM EDTA, 50 mM N-acetyl L-cysteine, 0.04 mMZn-acetate, 0.08% trehalose) and the mixture was incubated for 1 hr at42° C.

[0069] The amplification reactions were pooled for each patient (50,Ifrom each of tubes 1 and 2) and the amplified products were labeledusing previously described methods (PCT No. WO 99/65926 andPCT/IB99/02073). Briefly, the amplification products were mixed withlabeling reagent (final concentration of 60 mM MnCl₂, 6 mM imidazole, 2mM bromofluorescein in 150,μl total volume) and incubated at 60° C. for30 min, and then 15 μl of 0.5 M EDTA was added. Labeled nucleic acidswere purified using standard column chromatography (QIAVAC™, Qiagen, SA,Courtaboeuf, France) and eluted in a 100 μl volume which was mixed with400 μl of hybridization buffer (0.06 M HEPES, pH 7.5, 0.9 M NaCl, 3 Mbetaine, 5 mM DTAB, 500 ,g/ml salmon sperm DNA, 0.06% antifoam, 5×10⁻⁴nmol/test of control oligonucleotide). The mixture was incubated with anarray of immobilized DNA probes for 30 min at 35° C. (DNA-CHIP™ in aFluidic Station, Affymetrix, Inc., Santa Clara, Calif.; Lipshutz et al.,BioFeature 3:442-447(1995); U.S. Pat. No. 5,744,305). Unbound materialwas washed from the array twice (using a HEPES, NaCl, TRITON® X-102solution) and the hybridization results on the array were detected todetermine the relevant HIV-1 sequences present in the patient samples(Lipshutz et al., BioFeature 3:442-447(1995); PCT No. WO 95/11995). Thissystem can detect 1005 polymorphic HIV-1 gag and pol sequences covering180 mutations associated with drug-resistance (drug resistance codonsdetermined by French Agence Nationale de la Recherche sur le SIDA). Forthe detection step, the fluorescence intensity was measured for eachprobe site on the array (using an Affymetrix confocal laser reader), andan algorithm (Affymetrix GENECHIP™ software) was used to determine thebase corresponding to the most intense signal which was used todetermine the amino acid(s) likely to be present for a the codon. Thepredicted amino acids at selected positions obtained using this methodfor each of the three patient samples is shown, using the single letteramino acid code, in Table 5 for the RT codons and in Table 6 forProtease codons (“Chip” columns). For comparison, the HIV-1 sequence foreach patient sample was determined independently (using standardsequencing methods following RT-PCR as described in U.S. Pat. No.5,795,722), and the predicted amino acids for each of the codonscorresponding to those determined by hybridization analysis are shown inthe tables (“Seq” columns). For each reported position, the wild-type(non-drug-resistance) amino acid is shown in the first column (“WT &position”); “nd” means not determined.

[0070] These results show that the amplified HIV-1 sequences hybridizedto the DNA probes provided information on mutations affecting aminoacids associated with drug resistance (mutations shown in bold), andthat the hybridization results generally agreed with the predicted aminoacids determined by sequencing for 135 analyzed codons (98.5%). In twocases, probe hybridization detected a mutant sequence whereas DNAsequencing detected a mixture of wild-type and mutant sequences.Detection of the mutant, however, is clinically important for diagnosis.TABLE 5 RT Codons Predicted from Amplified HIV-1 RNA from Three PatientsPatient 1 Patient 2 Patient 3 WT & Position Chip Seq Chip Seq Chip SeqM41 M M L L L L A62 A A A A A A K65 K K K K nd K D67 D D N N N N S68 S SS S S S T69 T T T T T T K70 K K K K R R L74 L L L L V V V75 V V V V V VF77 F F F F F F A98 A A A A A A L100 L L L L L L K101 K K E E K K K103 KK K K R R V106 V V V V V V V108 V V V V V V Y115 Y Y Y Y Y Y F116 F F FF F F Q151 Q Q Q Q Q Q Y181 Y Y Y Y Y Y M184 V V V V M M Y188 Y Y Y Y YY G190 G G G G A A L210 L L L L W W T215 T T Y Y Y Y K219 K K Q Q E EP225 P P P P P P P236 P P P P P P

[0071] TABLE 6 Protease Codons Predicted from Amplified HIV-1 RNA fromThree Patients Patient 1 Patient 2 Patient 3 WT & Position Chip Seq ChipSeq Chip Seq L10 L L I L/I L L K20 K/I/M K/I/M K K R R D30 D D nd D D DM36 M M M M I I M46 M M nd M M M I47 I I I I I I G49 G G G G G G I50 I II I I I I54 I I I I I I L63 P P P P P P A71 A A A A A I G73 S G/S S S GG V77 I I I I V V V82 V V V V V V I84 I I I I V V N88 N N N N N N L90 MM M M M M

[0072] In other experiments, amplification of HIV-1 target sequences wassimilarly performed to produce detectable products using the followingcombinations of amplification oligomers: for Gag1 (SEQ ID NO:33 and SEQID NO:59), for Gag2 (SEQ ID NO:36 and SEQ ID NO:60), for Prt (SEQ IDNO:37 or SEQ ID NO:38 and SEQ ID NO:19, SEQ ID NO:61 or SEQ ID NO:62),for RT1 (SEQ ID NO:39 or SEQ ID NO:40 and SEQ ID NO:20, SEQ ID NO:63,SEQ ID NO:64 or SEQ ID NO:65), for RT3 (SEQ ID NO:42 or SEQ ID NO:43 andSEQ ID NO:66), and for RT4 (SEQ ID NO:44 or SEQ ID NO:45 and SEQ IDNO:67 or SEQ ID NO:68).

Example 5 PCR Amplification and Detection of Amplified HIV-1 Proteaseand RT1 Target Sequences

[0073] In this example, HIV-1 target sequences (Prt and RT1) wereamplified using the reverse transcriptase-polymerase chain reaction(RT-PCR) and the amplified sequences were then detected by using the DNAchip analysis as described in the previous example, compared to standardDNA sequencing. Viral RNA was purified from tissue culture supernatantof 92BR020 isolate (from The National Institutes of Health, Bethesda,Md.) using standard RNA purification methods (QIAMP™ Viral RNA Mini Kit,Qiagen, SA, Courtaboeuf, France) to produce an eluate containing 5×10⁵copies/ml. RT-PCR was performed using standard methods (ACCESS™ RT-PCRKit, Promega, Madison, Wis.) using the primers described in the previousexample for amplification of the Prt and RT1 target sequences (for Prt,SEQ ID NO:37 and SEQ ID NO:19; for RT1, SEQ ID NO:41 and SEQ ID NO:20),performed in separate tubes. After amplification, the results werechecked using electrophoresis through a 1.5% agarose gel stained withethidium bromide, relative to standard molecular size markers. A band ofamplified nucleic acid of the expected size was seen for RT1 for tubescontaining 5 copies/ml of target and for Protease for tubes containing500 copies/ml of target.

[0074] Following amplification, an aliquot from each tube was sequencedusing standard DNA sequencing methods. For hybridization to a DNA probearray on a chip, 10 μl of the amplified material was used as a templatefor transcription using T7 RNA polymerase in standard procedures (AMBIONMEGASCRIPT™ T7 kit, Clinisciences, Montrouge, France). Followingtranscription, 8 μl of RNA products were labeled with bromofluoresceinsubstantially as described in the previous example. The predicted aminoacid results for various positions of the Prt and RT1 amplifiedsequences are shown in Table 7, presented as described in the previousexample.

[0075] These results show that the amplification oligomers can produceamplicons by using another amplification method (RT-PCR) and that theproducts, or transcripts produced from the amplification products,provide sequence information relevant to mutations by hybridization to aprobe array and by sequencing. The results were in generally inagreement (91.1%) for the 45 codons analyzed. TABLE 7 Predicted AminoAcids from Protease and RT1 Amplified Sequences 92BR020 92BR020 WT &Position Chip Seq Protease L10 L L K20 nd K D30 D D M36 I I M46 M M I47I I G48 G G I50 I I I54 I I L63 L L A71 A A G73 G G V77 V V V82 V V I84I I N88 nd N L90 L L RT1 M41 M M A62 A A K66 K K D67 D D S68 S S T69 T TK70 K K L74 nd L V75 V V F77 F F A98 A A L100 L L K101 K K K103 K K V106nd V V108 V V Y115 Y Y F116 F F Q151 Q Q Y181 Y Y M184 M M Y188 Y Y G190G G

[0076] The invention is defined by the claims that follow and includesall legally equivalent embodiments of the claimed invention.

1 70 1 29 DNA Artificial Sequence Description of Artificial Sequence T7promoter sequence 1 gaaatttaat acgactcact atagggaga 29 2 37 DNAArtificial Sequence Description of Artificial Sequence T7 promotersequence 2 gaaattaata cgactcacta tagggagacc acattga 37 3 32 DNAArtificial Sequence Description of Artificial Sequence T7 promotersequence 3 aatttaatac gactcactat agggagacca ca 32 4 33 DNA ArtificialSequence Description of Artificial Sequence T7 promoter sequence 4gaaattaata cgactcacta tagggagacc aca 33 5 50 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide primer for Gag targetsequence 5 gaaatttaat acgactcact atagggagag tggctccttc tgataatgct 50 654 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide primer for Gag target sequence 6 gaaattaata cgactcactatagggagacc acattgatgc ccttcnttgc caca 54 7 52 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide primer for Proteasetarget sequence 7 aatttaatac gactcactat agggagacca cagccatcca ttcctggcttta 52 8 50 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide primer for Reverse Transcriptase target sequence 8aatttaatac gactcactat agggagacca cagctgccct atttctaagt 50 9 54 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotideprimer for Reverse Transcriptase target sequence 9 gaaattaata cgactcactatagggagacc acattgataa atttgatatg tcca 54 10 55 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide primer for ReverseTranscriptase target sequence 10 gaaattaata cgactcacta tagggagaccacactgttag ctgccccatc tacat 55 11 21 DNA Artificial Sequence Descriptionof Artificial Sequence Oligonucleotide primer for Gag target sequence 11gtggctcctt ctgataatgc t 21 12 17 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide primer for Gag target sequence 12tgcccttcnt tgccaca 17 13 20 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide primer for Protease target sequence13 gccatccatt cctggcttta 20 14 18 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide primer for Reverse Transcriptasetarget sequence 14 gctgccctat ttctaagt 18 15 21 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide primer for ReverseTranscriptase target sequence 15 ttgataaatt tgatatgtcc a 21 16 22 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotideprimer for Reverse Transcriptase target sequence 16 ctgttagctgccccatctac at 22 17 19 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide primer for Gag target sequence 17 gacaccaaggaagctttag 19 18 22 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide primer for Gag target sequence 18 tgggattaaataaaatagta ag 22 19 20 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide primer for Protease target sequence 19aaggaaggac accaaatgaa 20 20 20 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide primer for Reverse Transcriptasetarget sequence 20 gccattgaca gaagaaaaaa 20 21 17 DNA ArtificialSequence Description of Artificial Sequence Oligonucleotide primer forReverse Transcriptase target sequence 21 ttagaaatag ggcanca 17 22 22 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotideprimer for Reverse Transcriptase target sequence 22 acttaatagcagaaatacag aa 22 23 22 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide probe for hybridization to amplified Gagsequence 23 tcaggccata tcacctagaa ct 22 24 22 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide probe forhybridization to amplified Gag sequence of HIV-1 subtype A 24 ttgaatgcatgggtgaaggt aa 22 25 25 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide probe for hybridization to amplified Gagsequence 25 gttttggctg angcaatgag tcagg 25 26 18 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide probe forhybridization to amplified Protease sequence 26 gtaggaccta cacctgtc 1827 25 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide probe for hybridization to amplified ReverseTranscriptase sequence 27 gggcctgaaa atccatacaa tactc 25 28 22 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotideprobe for hybridization to amplified Reverse Transcriptase sequence 28agctggactg tcaatganat ac 22 29 25 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide probe for hybridization to amplifiedReverse Transcriptase sequence 29 gcatagtaat atggggaaag actcc 25 30 56DNA Artificial Sequence Description of Artificial Sequence Captureoligonucleotide 30 gctggaataa cttctgcttc tattttaaaa aaaaaaaaaaaaaaaaaaaa aaaaaa 56 31 57 DNA Artificial Sequence Description ofArtificial Sequence Capture oligonucleotide 31 tctgctgtcc ctgtaataaacccgtttaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 57 32 55 DNA ArtificialSequence Description of Artificial Sequence Capture oligonucleotide 32actgacgctc tcgcacccat cttttaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 55 33 54DNA Artificial Sequence Description of Artificial SequenceOligonucleotide primer for Gag target sequence 33 gaaattaata cgactcactatagggagacc acagtggctc cttctgataa tgct 54 34 53 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide primer for Gag targetsequence 34 gaaattaata cgactcacta tagggagacc acatgatgcc cttcnttgcc aca53 35 53 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide primer for Gag target sequence 35 gaaattaata cgactcactatagggagacc acagggtggc tccttctgat aat 53 36 53 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide primer for Gag targetsequence 36 gaaattaata cgactcacta tagggagacc acatgatgcc cttctttgcc aca53 37 53 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide primer for Protease target sequence 37 gaaattaatacgactcacta tagggagacc acagccatcc attcctggct tta 53 38 53 DNA ArtificialSequence Description of Artificial Sequence Oligonucleotide primer forProtease target sequence 38 gaaattaata cgactcacta tagggagacc acaccatccattcctggctt taa 53 39 52 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide primer for Reverse Transcriptasetarget sequence 39 gaaattaata cgactcacta tagggagacc acagctgccctatttctaag tc 52 40 51 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide primer for Reverse Transcriptase targetsequence 40 aaattaatac gactcactat agggagacta tgctgcccta tttctaagtc a 5141 51 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide primer for Reverse Transcriptase target sequence 41gaaattaata cgactcacta tagggagacc acagctgccc tatttctaag t 51 42 53 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotideprimer for Reverse Transcriptase target sequence 42 gaaattaatacgactcacta tagggagacc acatcttgat aaatttgata tgt 53 43 52 DNA ArtificialSequence Description of Artificial Sequence Oligonucleotide primer forReverse Transcriptase target sequence 43 gaaattaata cgactcactatagggagacc acatttcctg ttttcagatt tt 52 44 55 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide primer for ReverseTranscriptase target sequence 44 gaaattaata cgactcacta tagggagaccacacctgtta gctgccccat ctaca 55 45 55 DNA Artificial Sequence Descriptionof Artificial Sequence Oligonucleotide primer for Reverse Transcriptasetarget sequence 45 gaaattaata cgactcacta tagggagacc acatccctgttagctgcccc atcta 55 46 21 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide primer for Gag target sequence 46gtggctcctt ctgataatgc t 21 47 20 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide primer for Gag target sequence 47gggtggctcc ttctgataat 20 48 20 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide primer for Gag target sequence 48tgatgccctt cnttgccaca 20 49 20 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide primer for Gag target sequence 49tgatgccctt ctttgccaca 20 50 20 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide primer for Protease target sequence50 gccatccatt cctggcttta 20 51 20 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide primer for Protease target sequence51 ccatccattc ctggctttaa 20 52 19 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide primer for Reverse Transcriptasetarget sequence 52 gctgccctat ttctaagtc 19 53 23 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide primer for ReverseTranscriptase target sequence 53 tatgctgccc tatttctaag tca 23 54 18 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotideprimer for Reverse Transcriptase target sequence 54 gctgccctat ttctaagt18 55 20 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide primer for Reverse Transcriptase target sequence 55tcttgataaa tttgatatgt 20 56 19 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide primer for Reverse Transcriptasetarget sequence 56 tttcctgttt tcagatttt 19 57 22 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide primer for ReverseTranscriptase target sequence 57 cctgttagct gccccatcta ca 22 58 22 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotideprimer for Reverse Transcriptase target sequence 58 tccctgttagctgccccatc ta 22 59 19 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide primer for Gag target sequence 59 gacaccaaggaagctntag 19 60 22 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide primer for Gag target sequence 60 tgggnttaaataaaatagta ag 22 61 20 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide primer for protease target sequence 61gaaggacacc aaatgaagga 20 62 20 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide primer for Protease target sequence62 aagganggac accaaatgaa 20 63 22 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide primer for Reverse Transcriptasetarget sequence 63 gaagaaaaaa taaaagcatt ag 22 64 19 DNA ArtificialSequence Description of Artificial Sequence Oligonucleotide primer forReverse Transcriptase target sequence 64 gccattgaca gaagaaaaa 19 65 20DNA Artificial Sequence Description of Artificial SequenceOligonucleotide primer for Reverse Transcriptase target sequence 65gccattgaca gaagagaaaa 20 66 22 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide primer for Reverse Transcriptasetarget sequence 66 ggcancatag ancaaaaata ga 22 67 24 DNA ArtificialSequence Description of Artificial Sequence Oligonucleotide primer forReverse Transcriptase target sequence 67 agacttaata gcagaaatac agaa 2468 22 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide primer for Reverse Transcriptase target sequence 68acttaatagc agaantacag aa 22 69 24 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide probe Gag target sequence 69gttttggctg agcaatgagt cagg 24 70 23 DNA Artificial Sequence Descriptionof Artificial Sequence Oligonucleotide probe Gag target sequence 70tggagaaaat tagtagattt cag 23

We claim:
 1. A nucleic acid oligomer for amplifying a nucleotidesequence of HIV-1, comprising a sequence selected from the groupconsisting of SEQ ID NO:5 to SEQ ID NO:22 and SEQ ID NO:33 to SEQ IDNO:68.
 2. A nucleic acid oligomer according to claim 1, wherein theoligomer nucleic acid backbone comprises one or more 2′-O-methoxylinkages, peptide nucleic acid linkages, phosphorothioate linkages,methylphosphonate linkages or any combination of these linkages.
 3. Anucleic acid oligomer according to claim 1, wherein the oligomer is apromoter-primer comprising a sequence selected from the group consistingof SEQ ID NO:5 to SEQ ID NO:10 and SEQ ID NO:33 to SEQ ID NO:45, whereina 5′ portion of the sequence includes a promoter sequence for T7 RNApolymerase.
 4. A mixture of nucleic acid oligomers according to claim 1,wherein the mixture comprises oligomers for amplifying a first gagsequence and having a nucleotide sequence selected from the groupconsisting of SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:33, SEQ ID NO:35, SEQID NO:46, SEQ ID NO:47, SEQ ID NO:17 and SEQ ID NO:59.
 5. A mixture ofnucleic acid oligomers according to claim 1, wherein the mixturecomprises oligomers for amplifying a second gag sequence and having anucleotide sequence selected from the group consisting of SEQ ID NO:6,SEQ ID NO:12, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:48, SEQ ID NO:49,SEQ ID NO:18, and SEQ ID NO:60.
 6. A mixture of nucleic acid oligomersaccording to claim 1, wherein the mixture comprises oligomers foramplifying a Protease sequence and having a nucleotide sequence selectedfrom the group consisting of SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:37,SEQ ID NO:38, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:19, SEQ ID NO:61,and SEQ ID NO:62.
 7. A mixture of nucleic acid oligomers according toclaim 1, wherein the mixture comprises oligomers for amplifying a firstreverse transcriptase (RT) sequence and having a nucleotide sequenceselected from the group consisting of SEQ ID NO:8, SEQ ID NO:14, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:52, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:20, SEQ ID NO:63, SEQ ID NO:64 and SEQ ID NO:65.
 8. Amixture of nucleic acid oligomers according to claim 1, wherein themixture comprises oligomers for amplifying a second RT sequence andhaving a nucleotide sequence selected from the group consisting of SEQID NO:9, SEQ ID NO:15, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:21, and SEQ ID NO:66.
 9. A mixture of nucleic acidoligomers according to claim 1, wherein the mixture comprises oligomersfor amplifying a third RT sequence and having a nucleotide sequenceselected from the group consisting of SEQ ID NO:10, SEQ ID NO:16, SEQ IDNO:44, SEQ ID NO:45, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:22, SEQ IDNO:67, and SEQ ID NO:68.
 10. A labeled oligonucleotide that specificallyhybridizes to an HIV-1 sequence derived from gag or pol sequences,having a base sequence selected from the group consisting of SEQ IDNO:23 to SEQ ID NO:29, and a label that results in a detectable signal.11. A labeled oligonucleotide according to claim 10, wherein theoligonucleotide includes in its nucleic acid backbone one or more2′-O-methoxy linkages, peptide nucleic acid linkages, phosphorothioatelinkages, methylphosphonate linkages or any combination these linkages.12. A labeled oligonucleotide according to claim 10, wherein the labelis a compound that produces a luminescent signal that can be detected ina homogeneous detection system.
 13. A labeled oligonucleotide accordingto claim 10, wherein the label is an acridinium ester (AE) compound andthe oligonucleotide hybridizes to an HIV-1 sequence derived from gagsequences and has a base sequence selected from the group consisting of:SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25.
 14. A labeledoligonucleotide according to claim 10, wherein the label is anacridinium ester (AE) compound and the oligonucleotide hybridizes to anHIV-1 sequence derived from pol sequences and has a base sequenceselected from the group consisting of: SEQ ID NO:26, SEQ ID NO:27, SEQID NO:28, and SEQ ID NO:29.
 15. A method of detecting HIV-1 in abiological sample, comprising the steps of: providing a biologicalsample containing HIV-1 nucleic acid; mixing the sample with two or moreamplification oligomers that specifically amplify at least one HIV-1target sequence contained within gag and pol sequences under conditionsthat allow amplification of nucleic acid, wherein the amplificationoligomers have sequences selected from the group consisting of: SEQ IDNO:5, SEQ ID NO:11, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:17, and SEQ ID NO:59 to amplify a first gag sequence;SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:48, SEQID NO:49, SEQ ID NO:18, and SEQ ID NO:60 to amplify a second gagsequence; SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:50, SEQ ID NO:51, SEQ ID NO:19, SEQ ID NO:61 and SEQ ID NO:62 toamplify a first pol sequence, which is a protease sequence; SEQ ID NO:8,SEQ ID NO:14, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:52,SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:20, SEQ ID NO:63, SEQ ID NO:64,and SEQ ID NO:65 to amplify a second pol sequence, which is a firstreverse transcriptase (RT) sequence; SEQ ID NO:9, SEQ ID NO:15, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:21, and SEQID NO:66 to amplify a third pol sequence, which is a second RT sequence;and SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:22, SEQ ID NO:67, and SEQ ID NO:68 toamplify a fourth pol sequence, which is a third RT sequence; or acombination of oligomers selected from these groups that allowsamplification of at least one gag sequence and at least pol sequence;amplifying the target sequence to produce an amplified nucleic acidproduct; and detecting the presence of the amplified nucleic acidproduct.
 16. The method of claim 15, wherein the amplifying step uses atranscription-mediated amplification method which is conducted insubstantially isothermal conditions.
 17. The method of claim 15, whereinthe detecting step uses: a labeled oligomer having the sequence of SEQID NO:23, SEQ ID NO:24 or SEQ ID NO:25, or a mixture of these oligomers,to hybridize specifically to the amplified nucleic acid produced from agag sequence; a labeled oligomer having the sequence of SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29, or a mixture of theseoligomers, to hybridize specifically to the amplified nucleic acidproduced from a pol sequence; or a mixture of at least two labeledoligomers, wherein the mixture comprises one or more first labeledoligomers selected from the group consisting of SEQ ID NO:23, SEQ IDNO:24, and SEQ ID NO:25, and one or more second labeled oligomersselected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, and SEQ ID NO:29 to hybridize specifically to the amplifiednucleic acid produced from at least one gag and at least one polsequence.
 18. The method of claim 15, wherein the detecting step detectshybridization of the amplified nucleic acid to an array of nucleic acidprobes.
 19. The method of claim 15, further comprising the step ofcontacting the sample containing HIV-1 nucleic acid with at least onecapture oligomer having a sequence that hybridizes specifically to theHIV-1 nucleic acid, thus forming a hybridization complex that includesthe HIV-1 nucleic acid and separating the hybridization complex fromother sample components.